Introduction

Access to formalin-fixed, paraffin-embedded (FFPE) tissue is relatively easy, as it is evaluated, preserved, and stored in routine clinical diagnostics. FFPE tissue sample preparation for proteomic analysis, however, is extremely challenging and harsh conditions are required to remove cross-links and paraffin for efficient protein extraction. By combining the BeatBox® tissue homogenizer with an optimized iST sample preparation workflow, it became possible to process up to 96 samples of FFPE-curls per working day easily, efficiently, and reproducibly without using toxic xylene-based deparaffinization, opening up a new avenue for retrospective proteomic studies.

Proteomic labs typically receive FFPE tissue in various formats and amounts. To address this issue and to further increase workflow flexibility, we now aimed to optimize and adapt the workflow to higher FFPE input amounts.

Methods

FFPE samples (full curls, non-deparaffinized) from mouse tissues (cardiac muscle, kidney, and liver) and matching snap-frozen tissue samples were processed in plate or tube format using BeatBox homogenization coupled to iST sample preparation. For FFPE samples, an optimized workflow was established: FFPE curls were homogenized in the BeatBox (10 minutes), followed by an one-hour heat incubation, to de-crosslink, extract, reduce and alkylate proteins. Applying the iST sample preparation protocol, tryptic digestion was followed by an optimized peptide clean-up with an additional washing step to remove last traces of paraffin. To extend the workflow for larger FFPE input material, the workflow was adjusted accordingly. Peptides were analyzed on a nano-LC coupled to a timsTOF mass spectrometer. Raw files were analyzed using Bruker ProteoScapeTM.

Results

This novel xylene-free BeatBox-iST workflow was applied to FFPE curls in plate or tube format and compared to a traditional workflow with xylene-based deparaffinization and bead-based sonication. For all tested tissue types, BeatBox homogenization outperformed bead-based sonication, revealing an increase in protein IDs of >10%, with a remarkable increase of 43% for mouse cardiac muscle tissue. Notably, the BeatBox-iST protocol achieved excellent intra- and interday repeatability with median CVs of <10% (n=4). Besides, it could be ensured that paraffin was completely removed from FFPE samples and no contamination was introduced into the LC-MS instrumentation.

Comparing FFPE tissues with the corresponding fresh-frozen tissues revealed more than 10´000 proteins for both sample formats with up to 73% shared proteins and a similar dynamic range, confirming the superior performance of the FFPE BeatBox-iST workflow.

Conclusion

This BeatBox-iST workflow simplifies large-scale retrospective proteomic studies by providing a xylene-free, robust and high-throughput solution for FFPE tissue samples and can now also be applied to larger FFPE input material.